Effects of PTHrP on chondrocytes of sika deer antler.

Parathyroid-hormone-related peptide (PTHrP) is an important regulator of chondrocyte differentiation in growth plates but little is known about its role in deer antler cartilage. The aim of the present study was to use the deer antler as a model to determine the possible role of PTHrP in regulating chondrocyte differentiation of deer antler. PTHrP and its receptor PTH1R mRNA were highly expressed in the perichondrium and cartilage of sika deer antler, as shown by in situ hybridization. Chondrocytes of deer antler were identified by toluidine blue staining of glycosaminoglycan and immunocytochemical staining of type II collagen (Col II). Treatment with PTHrP (1-34) reduced the expression of prehypertrophic chondrocyte marker Col IX and hypertrophic chondrocyte marker Col X. In order to confirm the mechanism of action of PTHrP, we initially examined the expression of cyclin D1, Bcl-2 and runt-related transcription factor 2 (Runx2) in sika deer antler by in situ hybridization and found that cyclin D1, Runx2 and Bcl-2 mRNA were also expressed in antler chondrocytes. Exogenous PTHrP induced the expression of cyclin D1 and Bcl-2 mRNA by various signalling pathways, whereas it inhibited Runx2 expression through PKA, p38MAPK, MEK and PI3K signalling pathways. Thus, PTHrP might promote the proliferation of antler chondrocytes and prevent their differentiation; it might furthermore influence the growth and development of sika deer antler.

Differential expression and regulation of Cryab in mouse uterus during preimplantation period.

The aim of this study was to examine the expression and regulation of the crystallin, alpha B (Cryab) gene in mouse uterus during the peri-implantation period by in situ hybridization and real-time PCR. There was no detectable Cryab mRNA signal on days 1-4 of pregnancy. On day 5 of pregnancy when embryo implanted, a high level of Cryab mRNA signal was found in the subluminal stroma surrounding the implanting blastocyst. On days 6-8, Cryab mRNA was strongly expressed in the primary decidua. By real-time PCR, a high level of Cryab expression was detected on days 7 and 8 of pregnancy, although Cryab expression was seen from days 1 to 8. Under in vivo and in vitro artificial decidualization, Cryab expression was significantly elevated. Compared with the progesterone-primed delayed implantation uterus, a high level of Cryab mRNA expression was observed in estrogen-activated implantation uterus. In the uterine stromal cells, cAMP, estrogen, and progesterone could induce the expression of Cryab gene. In the ovariectomized mouse uterus, estrogen could also induce the expression of Cryab while progesterone inhibited its expression. Our data suggest that Cryab may play an important role during mouse embryo implantation and decidualization and that estrogen and progesterone can regulate the expression of Cryab gene.

Effects of PTHrP on expression of MMP9 and MMP13 in sika deer antler chondrocytes.

Deer antlers are the only mammalian appendages to display an annual cycle of full regeneration. However, little is known about the molecular mechanisms of antler regeneration. Our previous study has demonstrated that parathyroid hormone-related peptide (PTHrP) can promote proliferation of antler chondrocytes and inhibit its differentiation, but the mechanism underlying such regulation is not fully understood. We have determined the role of PTHrP on the mRNA expression of matrix metalloproteinase-9 (MMP9) and MMP13 in the antler chondrocytes. The possible pathways that transduce PTHrP effects were examined. In situ hybridization showed that MMP9 and MMP13 were mainly localized in the dermal fibroblasts, perichondrium, and cartilage in the sika deer antler, of which MMP9 and MMP13 were highly expressed in the chondrocytes. Exogenous PTHrP could inhibit the expression of MMP9 and MMP13 in the antler chondrocytes. The inhibitory effect of PTHrP on MMP9 was abolished by JNK inhibitor, SP600125, while P38MAPK inhibitor SB203850 and PKC inhibitor GF109203X could rescue the inhibitory effect of PTHrP on MMP13. The results suggest that PTHrP can inhibit MMP9 expression by JNK signaling pathway and MMP13 expression by p38MAPK and PKC signaling pathways in the antler chondrocytes. Thus PTHrP is involved in the control of antler chondrocytes maturation and cartilage matrix degradation.

Differential expression and regulation of angiopoietin-3 in mouse uterus during preimplantation period.

Angiogenesis is necessary for successful implantation and decidualization. This study was to investigate the differential expression of angiopoietin-3 (Ang-3) in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). There was no detectable Ang-3 mRNA signal on days 1-5 of pregnancy by in situ hybridization. On day 6 of pregnancy, a low level of Ang-3 mRNA signal was seen in the primary decidua. Ang-3 mRNA expression gradually increased on days 7 and 8 of pregnancy along with the development of decidua, and its expression scope was also expanded. The RT-PCR result indicated that Ang-3 mRNA expression was low on days 1-4 of pregnancy. On day 5, as embryo implanted, Ang-3 mRNA was highly expressed in mouse uterus, and the expression gradually increased on days 6-8 of pregnancy, with peak level on day 8 of pregnancy. Similarly, Ang-3 mRNA was also strongly expressed in decidualized cells under artificial decidualization. Compared with the delayed uterus, a high level of Ang-3 mRNA expression was detected in activated implantation uterus by RT-PCR. In the ovariectomized mouse uterus, Ang-3 mRNA expression increased and reached the highest level at 12 hr after injection of estrogen, progesterone, and estrogen plus progesterone, respectively. These results suggest that Ang-3 may play an important role during the process of mouse decidualization. Both estrogen and progesterone can induce the expression of Ang-3 in ovariectomized mouse uterus.

Expression and regulation of Runx3 in mouse uterus during the peri-implantation period.

The aim of this study was to investigate the differential expression and regulation of Runt-related transcription factor 3 (Runx3) in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization. There was a low level of the Runx3 mRNA expression in the mouse uterus on days 1-4 of pregnancy. On day 5 when embryo implanted, Runx3 mRNA signal was obviously observed in the stromal cells surrounding the implanting blastocyst. From day 6 to 8 of pregnancy, Runx3 mRNA was highly expressed in the decidual cells and mesometrial decidual beds. Similarly, Runx3 mRNA was strongly expressed in decidualized cells under artificial decidualization. Compared with the delayed uterus, a high level of Runx3 mRNA signal was detected in the uterus with activated implantation. In the ovariectomized mouse uterus, estrogen could induce the expression of Runx3, while progesterone had no effects. These results suggest that Runx3 may play an important role during mouse implantation and decidualization. Estrogen can induce the expression of Runx3 in the ovariectomized mouse uterus.